The synthetic modification of proteins plays an essential role in the fields of biomaterials science and chemical biology. Conjugation of synthetic molecules to proteins enables the incredible diversity of protein structure and function to be harnessed in new protein-based materials. Protein bioconjugates have been used for a wide array of applications from the investigation of biological function to use in new biomaterials such as targeted therapeutics and biocatalysts. The synthesis of these materials requires well-defined protein bioconjugation reactions. However, a significant challenge is that these reactions must take place in water, at ambient temperature, near neutral pH and in the presence of a wide array of functional groups.
While many methods for the modification of native and artificial amino acids exist, they often require long reaction times, lengthy syntheses of reactive substrates, or lack specificity and selectivity. To address these issues, a suite of bioconjugation reactions that utilize ortho-aminophenols was developed. The oxidative coupling of non-canonical aniline residues with o-aminophenol substrates was developed to enable use with a wide range of protein substrates, including those containing free cysteines and glycosylation. In the course of these studies, it was discovered that under certain conditions aminophenols were reactive with native residues on protein substrates in addition to artificial aniline moieties. The reactive native residues were identified and oxidative coupling of o-aminophenols with the N-terminus was optimized to achieve high levels of modification on peptide and protein substrates. This new reactivity dramatically expands the bioconjugation reaction toolkit, enabling fast, site-selective modification in a single step using low concentrations of reagent.
In addition to the development of methods for protein modification, the use of modified proteins for targeted delivery and biocatalysis was explored. Two methods for the self-assembly of protein biocatalysts were explored, both relying on the ability of block copolymers to direct self-assembly. First, site-specific functionalization of cytochrome P450 BM3, a highly pliable enzyme, with poly(N-isopropylacrylamide) to create a diblock copolymer generates a material that can be easily processed to create a self-assembled heterogeneous biocatalyst. The self-assembly of the bioconjugate in concentrated solutions was characterized by small-angle x-ray scattering (SAXS) and depolarized light scattering (DPLS). The thin film nanostructure was also investigated by grazing-incidence small-angle x-ray scattering (GISAXS). Catalytic activity of the thin films for C-H oxidation was measured using a variety of substrates. An additional method for controlling the self-assembly of proteins was probed. Non-specific modification of proteins to vary their surface charge was also examined as a means for directing protein self-assembly. Using model proteins and lysine succinylation, a panel of proteins with varying charge density was synthesized. The ability of these proteins to form complex coacervates with oppositely charged polyelectrolytes was studied in order to identify parameters that governed complex coacervation of proteins. The fundamental parameters uncovered allow any proteins of interest to be successfully modified to enable complex coacervation. These findings were applied to the synthesis of complex coacervate core micelles with protein cores. Encapsulation of enzymes in these ionic micelles enables their use as biocatalysts and future work will investigate the use of these micelles for protein drug delivery.
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